Campylobacter spp, generally food-borne, are the leading cause of gastroenteritis worldwide, surpassing the number of cases of Salmonella and Shigella combined. Campylobacter spp. are also associated with the development of Guillain-Barre syndrome, which is the most common cause of acute neuromuscular paralysis. In addition, since Campylobacter could be easily acquired and spread through our food supply, it constitutes a potential bioterrorism threat. Currently, no vaccine is available against Campylobacter infection, and despite an intensive research effort to understand Campylobacter pathophysiology, conclusions on the exact mechanism of infection are extremely difficult to draw. C. jejuni is adapted to survive both in the environment (mainly water and milk) and in its host organisms (mammals and birds). Upon entrance into the human host, Campylobacter must survive in the intestinal tract, either as a free bacterium in the mucus layer or intracellularly in gut epithelial ceils. To colonize the intestinal tract, C. jejuni must successfully transit through the gastric acid barrier of the stomach to the more alkaline environment of the intestine. While up to 500 commensal species as well as other food-borne pathogens must similarly surmount these host barriers and adapt to the gut environment, very little is known about this process. This proposal focuses on the characterization of Campylobacter jejuni colonization and virulence factors. This proposal is based on the following hypothesis: there are numerous genes expressed in rivo that are influenced by environmental factors, and several of these genes are required for gut colonization and ultimately disease development. C. jejuni colonization and virulence determinants will be identified by in vitro and in vivo survival analysis of insertional mutants using DNA microarray. First, a functional genomic tool will be developed to identify conditionally essential genes in C. jejuni, and validated to examine the mechanism of C. jejuni survival to acid stress. Second, this functional genomic tool will be used to characterize the interactions of C. jejuni with the host gastrointestinal tract using the newborn piglet as an animal model of human infection. Finally, the role of the colonization and virulence determinants in disease and Campylobacter physiology will be assessed using a battery of in vitro biological assays. The identification of these Campylobacter determinants could significantly contribute to the development of more effective methods to diagnose, manage and ultimately prevent Campylobacter infections.